Activation of the Constitutive Androstane Receptor Inhibits Leukocyte Adhesiveness to Dysfunctional Endothelium

Leukocyte cell recruitment into the vascular subendothelium constitutes an early event in the atherogenic process. As the effect of the constitutive androstane receptor (CAR) on leukocyte recruitment and endothelial dysfunction is poorly understood, this study investigated whether the role of CAR activation can affect this response and the underlying mechanisms involved. Under physiological flow conditions, TNFα-induced endothelial adhesion of human leukocyte cells was concentration-dependently inhibited by preincubation of human umbilical arterial endothelial cells with the selective human CAR ligand CITCO. CAR agonism also prevented TNFα induced VCAM-1 expression, as well as MCP-1/CCL-2 and RANTES/CCL-5 release in endothelial cells. Suppression of CAR expression with a small interfering RNA abrogated the inhibitory effects of CITCO on these responses. Furthermore, CITCO increased interaction of CAR with Retinoid X Receptor (RXR) and reduced TNFα-induced p38-MAPK/NF-κB activation. In vivo, using intravital microscopy in the mouse cremasteric microcirculation treatment with the selective mouse CAR ligand TCPOBOP inhibited TNFα-induced leukocyte rolling flux, adhesion, and emigration and decreased VCAM-1 in endothelium. These results reveal that CAR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps in the leukocyte recruitment cascade. Therefore, CAR agonists may constitute a new therapeutic tool in controlling cardiovascular disease-associated inflammatory processes.     

The Aryl Hydrocarbon Receptor Undergoes Chaperone-Mediated Autophagy in Triple-Negative Breast Cancer Cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule expressed in many cell types, including triple-negative and non-triple-negative breast cancer cells. It affects breast cancer growth and crosstalk with estrogen receptor signaling. Normally, this receptor is degraded shortly after ligand activation via the 26S proteasome. Here, we report that AHR undergoes chaperone-mediated autophagy in MDA-MB-468 triple-negative breast cancer cells. This lysosomal degradation of AHR exhibits the following characteristics: (1) it is triggered by 6 amino-nicotinamide, starvation, and piperazinylpyrimidine compound Q18; (2) it is not observed in non-triple-negative breast cancer cells (MCF-7, T47D, and MDA-MB-361); (3) it can be inhibited by progesterone receptor B but not estrogen receptor alpha; (4) it can be reversed by chloroquine but not MG132; (5) it requires LAMP2A; and (6) it involves AHR-HSC70 and AHR-LAMP2A interactions. The NEKFF sequence localized at amino acid 558 of human AHR appears to be a KFERQ-like motif of chaperone-mediated autophagy, responsible for the LAMP2A-mediated AHR protein degradation.     

Aryl Hydrocarbon Receptor Repressor and TiPARP (ARTD14) Use Similar,but also Distinct Mechanisms to Repress Aryl Hydrocarbon Receptor Signaling

The aryl hydrocarbon receptor (AHR) regulates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AHR repressor (AHRR) is an AHR target gene and functions as a ligand-induced repressor of AHR; however, its mechanism of inhibition is controversial. Recently, we reported that TCDD-inducible poly (ADP-ribose) polymerase (TiPARP; ARTD14) also acts as a repressor of AHR, representing a new player in the mechanism of AHR action. Here we compared the ability of AHRR- and TiPARP-mediated inhibition of AHR activity. TCDD increased AHRR mRNA levels and recruitment of AHRR to cytochrome P450 1A1 (CYP1A1) in MCF7 cells. Knockdown of TiPARP, but not AHRR, increased TCDD-induced CYP1A1 mRNA and AHR protein levels. Similarly, immortalized TiPARP^−/− mouse embryonic fibroblasts (MEFs) and AHRR^−/− MEFs exhibited enhanced AHR transactivation. However, unlike TiPARP^−/− MEFs, AHRR^−/− MEFs did not exhibit increased AHR protein levels. Overexpression of TiPARP in AHRR^−/− MEFs or AHRRΔ8, the active isoform of AHRR, in TiPARP^−/− MEFs reduced TCDD-induced CYP1A1 mRNA levels, suggesting that they independently repress AHR. GFP-AHRRΔ8 and GFP-TiPARP expressed as small diffuse nuclear foci in MCF7 and HuH7 cells. GFP-AHRRΔ8_Δ1-49, which lacks its putative nuclear localization signal, localized to both the nucleus and the cytoplasm, while the GFP-AHRRΔ8_Δ1-100 mutant localized predominantly in large cytoplasmic foci. Neither GFP-AHRRΔ8_Δ1-49 nor GFP-AHRRΔ8_Δ1-100 repressed AHR. Taken together, AHRR and TiPARP repress AHR transactivation by similar, but also different mechanisms.     

Epigenetic Determinants of CYP1A1 Induction by the Aryl Hydrocarbon Receptor Agonist 3,3',4,4',5-Pentachlorobiphenyl (PCB 126)

Many enzymes involved in xenobiotic metabolism, including cytochrome P450 (CYP) 1A1, are regulated by the aryl hydrocarbon receptor (AhR). 3,3'',4,4'',5-Pentachlorobiphenyl (PCB 126) is a potent ligand for AhR and can thus induce the expression of CYP1A1. Interestingly, we observed that human carcinoma cell lines derived from different types of epithelial cells displayed divergent degrees of CYP1A1 induction after exposure to PCB 126. Since epigenetic mechanisms are known to be involved in cell type-specific gene expression, we sought to assess the epigenetic determinants of CYP1A1 induction in these carcinoma cell lines. In contrast to HepG2 hepatocarcinoma cells, HeLa cervical carcinoma cells showed significantly lower levels of CYP1A1 mRNA expression following PCB 126 exposure. Our results show that the two cell lines maintained differences in the chromatin architecture along the CYP1A1 promoter region. Furthermore, treatment with the epigenetic modifiers, trichostatin A (TSA) and 5-aza-2''-deoxycytidine (5-Aza-dC), significantly increased the expression of CYP1A1 after PCB 126 treatment in HeLa cells. However, we did not observe apparent differences in methylation levels or specific location of CpG DNA methylation between the two cell lines in the analyzed CYP1A1 promoter region. Taken together, our findings suggest that the differences in CYP1A1 expression between HepG2 and HeLa cells are due to differences in the chromatin architecture of the CYP1A1 promoter and thus establish a role of epigenetic regulation in cell-specific CYP1A1 expression.     

Baicalin Protects Mice from Aristolochic Acid I-Induced Kidney Injury by Induction of CYP1A through the Aromatic Hydrocarbon Receptor

Exposure to aristolochic acid I (AAI) can lead to aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and urothelial cancer. The induction of hepatic CYP1A, especially CYP1A2, was considered to detoxify AAI so as to reduce its nephrotoxicity. We previously found that baicalin had the strong ability to induce CYP1A2 expression; therefore in this study, we examined the effects of baicalin on AAI toxicity, metabolism and disposition, as well as investigated the underlying mechanisms. Our toxicological studies showed that baicalin reduced the levels of blood urea nitrogen (BUN) and creatinine (CRE) in AAI-treated mice and attenuated renal injury induced by AAI. Pharmacokinetic analysis demonstrated that baicalin markedly decreased AUC of AAI in plasma and the content of AAI in liver and kidney. CYP1A induction assays showed that baicalin exposure significantly increased the hepatic expression of CYP1A1/2, which was completely abolished by inhibitors of the Aromatic hydrocarbon receptor (AhR), 3ʹ,4ʹ-dimethoxyflavone and resveratrol, in vitro and in vivo, respectively. Moreover, the luciferase assays revealed that baicalin significantly increased the luciferase activity of the reporter gene incorporated with the Xenobiotic response elements recognized by AhR. In summary, baicalin significantly reduced the disposition of AAI and ameliorated AAI-induced kidney toxicity through AhR-dependent CYP1A1/2 induction in the liver.     

Aryl Hydrocarbon Receptor Defect Attenuates Mitogen-Activated Signaling through Leucine-Rich Repeats and Immunoglobulin-like Domains 1 (LRIG1)-Dependent EGFR Degradation

Aryl hydrocarbon receptor (AHR) genomic pathway has been well-characterized in a number of respiratory diseases. In addition, the cytoplasmic AHR protein may act as an adaptor of E3 ubiquitin ligase. In this study, the physiological functions of AHR that regulate cell proliferation were explored using the CRISPR/Cas9 system. The doubling-time of the AHR-KO clones of A549 and BEAS-2B was observed to be prolonged. The attenuation of proliferation potential was strongly associated with either the induction of p27^Kip1 or the impairment in mitogenic signal transduction driven by the epidermal growth factor (EGF) and EGF receptor (EGFR). We found that the leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1), a repressor of EGFR, was induced in the absence of AHR in vitro and in vivo. The LRIG1 tends to degrade via a proteasome dependent manner by interacting with AHR in wild-type cells. Either LRIG1 or a disintegrin and metalloprotease 17 (ADAM17) were accumulated in AHR-defective cells, consequently accelerating the degradation of EGFR, and attenuating the response to mitogenic stimulation. We also affirmed low AHR but high LRIG1 levels in lung tissues of chronic obstructive pulmonary disease (COPD) patients. This might partially elucidate the sluggish tissue repairment and developing inflammation in COPD patients.     

Monoacylglycerols Activate Capsaicin Receptor, TRPV1

Transient receptor potential vanilloid subtype 1 (TRPV1) is known as capsaicin (CAP) receptor and activated by CAP. Activation of TRPV1 by CAP increases energy expenditure and thermogenesis in rodents or human. Therefore, TRPV1 may be target for energy expenditure enhancement and thermogenesis. To search for novel TRPV1 agonist, we screened 19 types of foods by using TRPV1-expressing HEK293 cells. TRPV1 was activated by hexane extract of wheat flour, and its functional compounds were 1-monoacylglycerols containing oleic, linoleic, and alpha-linolenic acids. Their potencies (EC50) were about 50 times larger than that of CAP and their efficacies (maximal response) were about half of that of CAP. TRPV1 was activated by 1-monoacylglycerols (MGs) having C18 and C20 unsaturated and C8-C12 saturated fatty acid (FA). Moreover, 2-MGs having C18 and C20 unsaturated FA acted on TRPV1 with the same potency. On the other hand, no activation of TRPV1 was induced by MGs having C16 and C18 saturated FA, di- or triacylglycerols of C18:1 FA. Pain-relating aversive responses were induced when TRPV1-activating 1-monoacylglycerols (50 mM) was administered subcutaneously into rat hind paw. These effects were inhibited by the co-injection of capsazepine (10 mM) which is a TRPV1 competitive antagonist. These results suggested that these 1-monoacylglycerols activate TRPV1 in vitro and in vivo.     

The up-regulation of hepatic acyl-coA oxidase and cytochrome P<Subscript>450</Subscript> 4A1 mRNA expression by dietary oxidized frying oil is comparable between male and female rats

We previously demonstrated that oxidized frying oil (OFO) activates peroxisome proliferator-activated receptor α (PPARα) and up-regulates hepatic acyl-CoA oxidase (ACO) and cytochrome P₄₅₀ 4A1 (CYP4A1) genes in male rats. As female rats were shown to be less responsive to some peroxisome proliferators (PP), this study compared the expression of a few PPARα target genes in male and female rats fed diets containing OFO. Male and female rats were fed a diet containing 20 g/100 g OFO (O diet) or fresh soybean oil (F diet) for 6 wk. Both male and female rats fed the O diet showed significantly higher liver weight, hepatic ACO and catalase activities, CYP4A protein, and expression of ACO and CYP4A1 mRNA (P<0.05) compared with their control groups. The mRNA expression of two other PPARα target genes, FA-binding protein and HMG-CoA synthase, were marginally increased by dietary OFO (P=0.0669 and 0,0521, respectively). Female rats fed the O diet had significantly lower CYP4A protein than male rats fed the same diet. The remaining OFO-induced effects were not significantly different between male and female rats fed the O diet. These results indicate that dietary OFO, unlike clofibrate or other PP, had minimal sexual dimorphic effect on the induction of hepatic PPARα target gene expression.     

Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.     

A Necessary Role for Increased Biglycan Expression during L1-Mediated Colon Cancer Progression

Aberrant activation of Wnt/β-catenin signaling and downstream β-catenin-TCF target genes is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin-like cell adhesion receptor L1CAM (L1) as a target of β-catenin-TCF transactivation in CRC cells. Overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis, and liver metastasis, and L1 is exclusively localized at invasive areas of human CRC tissue. Several genes are induced after L1 transfection into CRC cells by a mechanism involving the L1-ezrin-NF-κB pathway. We conducted a secretomic analysis of the proteins in the culture medium of L1-overexpressing CRC cells. We detected a highly increased level of biglycan, a small leucine-rich ECM component, and a signaling molecule. We found that induction of biglycan is required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis, and liver metastasis. The suppression of endogenous biglycan levels or a point mutation in the L1 ectodomain that regulates cell–cell adhesion mediated by L1 blocked the enhanced tumorigenic properties conferred by L1. The mechanism of biglycan induction by L1 involves the L1-NF-κB pathway. Blocking NF-κB signaling in L1 expressing cells suppressed the induction of biglycan and the tumorigenic properties conferred by L1. Biglycan expression was undetectable in the normal colonic mucosa, but expressed at highly increased levels in the tumor tissue, especially in the stroma. The therapeutic strategies to target biglycan expression might provide a useful approach for CRC treatment in L1-overexpressing tumors.     

Rapamycin Alleviates 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Induced Aggravated Dermatitis in Mice with Imiquimod-Induced Psoriasis-Like Dermatitis by Inducing Autophagy

Recently, the mTOR signaling has emerged as an important player in the pathogenesis of psoriasis. We previously found that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced psoriatic skin inflammation was related to the inhibition of autophagy in keratinocytes. However, the effects and detailed molecular mechanisms of the mTOR inhibitor rapamycin and TCDD on psoriasis in vivo remain to be elucidated. In this study, we aimed to evaluate the effects of rapamycin and TCDD on skin lesions in imiquimod (IMQ)-induced psoriasis using a mouse model. TCDD aggravated skin inflammation in an IMQ-induced psoriatic mouse model. Furthermore, TCDD increased the expression of aryl hydrocarbon receptor (AHR), CYP1A1, proinflammatory cytokines, oxidative stress markers (NADPH oxidase (Nox) 2, Nox4), and phosphorylated P65NF-ĸB, whereas the expression of autophagy-related factors and the antioxidant marker nuclear factor-erythroid 2-related factor 2 (NRF2) decreased. Rapamycin reduced the aggravated skin inflammation induced by TCDD and restored TCDD-induced autophagy suppression and the increase of AHR expression, oxidative stress, and inflammatory response in the skin lesions of a psoriatic mouse model. In conclusion, we demonstrated that rapamycin alleviates TCDD-induced aggravated dermatitis in mice with imiquimod-induced psoriasis-like dermatitis through AHR and autophagy modulation.     

Benzo[a]pyrene-Induced Genotoxicity in Rats Is Affected by Co-Exposure to Sudan I by Altering the Expression of Biotransformation Enzymes

The environmental pollutant benzo[a]pyrene (BaP) is a human carcinogen that reacts with DNA after metabolic activation catalysed by cytochromes P450 (CYP) 1A1 and 1B1 together with microsomal epoxide hydrolase. The azo dye Sudan I is a potent inducer of CYP1A1/2. Here, Wistar rats were either treated with single doses of BaP (150 mg/kg bw) or Sudan I (50 mg/kg bw) alone or with both compounds in combination to explore BaP-derived DNA adduct formation in vivo. Using ³²P-postlabelling, DNA adducts generated by BaP-7,8-dihydrodiol-9,10-epoxide were found in livers of rats treated with BaP alone or co-exposed to Sudan I. During co-exposure to Sudan I prior to BaP treatment, BaP-DNA adduct levels increased 2.1-fold in comparison to BaP treatment alone. Similarly, hepatic microsomes isolated from rats exposed to Sudan I prior to BaP treatment were also the most effective in generating DNA adducts in vitro with the activated metabolites BaP-7,8-dihydrodiol or BaP-9-ol as intermediates. DNA adduct formation correlated with changes in the expression and/or enzyme activities of CYP1A1, 1A2 and 1B1 in hepatic microsomes. Thus, BaP genotoxicity in rats in vivo appears to be related to the enhanced expression and/or activity of hepatic CYP1A1/2 and 1B1 caused by exposure of rats to the studied compounds. Our results indicate that the industrially employed azo dye Sudan I potentiates the genotoxicity of the human carcinogen BaP, and exposure to both substances at the same time seems to be hazardous to humans.     

Coal dust alters β-naphthoflavone-induced aryl hydrocarbon receptor nuclear translocation in alveolar type II cells

Background  

Many polycyclic aromatic hydrocarbons (PAHs) can cause DNA adducts and initiate carcinogenesis. Mixed exposures to coal dust (CD) and PAHs are common in occupational settings. In the CD and PAH-exposed lung, CD increases apoptosis and causes alveolar type II (AT-II) cell hyperplasia but reduces CYP1A1 induction. Inflammation, but not apoptosis, appears etiologically associated with reduced CYP1A1 induction in this mixed exposure model. Many AT-II cells in the CD-exposed lungs have no detectable CYP1A1 induction after PAH exposure. Although AT-II cells are a small subfraction of lung cells, they are believed to be a potential progenitor cell for some lung cancers. Because CYP1A1 is induced via ligand-mediated nuclear translocation of the aryl hydrocarbon receptor (AhR), we investigated the effect of CD on PAH-induced nuclear translocation of AhR in AT-II cells isolated from in vivo-exposed rats. Rats received CD or vehicle (saline) by intratracheal (IT) instillation. Three days before sacrifice, half of the rats in each group started daily intraperitoneal injections of the PAH, β-naphthoflavone (BNF).